We have cloned the cDNA encoding the second largest subunit of RNA polymerase I, termed RPA2, from mouse cells. The cDNA has a 3978-nucleotide open reading frame encoding a polypeptide of 1136 amino acids with a calculated molecular mass of 128 kDa. A sequence alignment of mouse RPA2 with the corresponding gene from Drosophila melanogaster and yeast reveals a much lower sequence similarity of this subunit of RNA polymerase I (Pol I) compared to the second largest subunit of other eukaryotic RNA polymerases. Four Pol I-specific regions, termed I alpha-I delta, are conserved in the N-terminal part of RPA2. The structural features of the different domains as well as the homology to essential functional domains found in other RNA polymerases are discussed.